Dendrites determine the contribution of after depolarization potentials (ADPs) to generation of repetitive action potentials in hypothalamic gonadotropin releasing-hormone (GnRH) neurons

The impact of structure in modulating synaptic signals originating in dendrites is widely recognized. In this study, we focused on the impact of dendrite morphology on a local spike generating mechanism which has been implicated in hormone secretion, the after depolarization potential (ADP). Using multi-compartmental models of hypothalamic GnRH neurons, we systematically truncated dendrite length and determined the consequence on ADP amplitude and repetitive firing. Decreasing the length of the dendrite significantly increased the amplitude of the ADP and increased repetitive firing. These effects were observed in dendrites both with and without active conductances suggesting they largely reflect passive characteristics of the dendrite. In order to test the findings of the model, we performed whole-cell recordings in GnRH neurons and elicited ADPs using current injection. During recordings, neurons were filled with biocytin so that we could determine dendritic and total projection (dendrite plus axon) length. Neurons exhibited ADPs and increasing ADP amplitude was associated with decreasing dendrite length, in keeping with the predictions of the models. Thus, despite the relatively simple morphology of the GnRH neuron’s dendrite, it can still exert a substantial impact on the final neuronal output. This work was supported by HD-45436 to KJS and by NCRR P20 RR16481 to Nigel Cooper.     

Regulation of Electrical Bursting in a Spatiotemporal Model of a GnRH Neuron

Gonadotropin-releasing hormone (GnRH) neurons are hypothalamic neurons that control the pulsatile release of GnRH that governs fertility and reproduction in mammals. The mechanisms underlying the pulsatile release of GnRH are not well understood. Some mathematical models have been developed previously to explain different aspects of these activities, such as the properties of burst action potential firing and their associated Ca²⁺ transients. These previous studies were based on experimental recordings taken from the soma of GnRH neurons. However, some research groups have shown that the dendrites of GnRH neurons play very important roles. In particular, it is now known that the site of action potential initiation in these neurons is often in the dendrite, over 100 μm from the soma. This raises an important question. Since some of the mechanisms for controlling the burst length and interburst interval are located in the soma, how can electrical bursting be controlled when initiated at a site located some distance from these controlling mechanisms? In order to answer this question, we construct a spatio-temporal mathematical model that includes both the soma and the dendrite. Our model shows that the diffusion coefficient for the spread of electrical potentials in the dendrite is large enough to coordinate burst firing of action potentials when the initiation site is located at some distance from the soma.     

Dendritic spikes and activity-dependent synaptic plasticity

Whereas the regenerative nature of action potential conduction in axons has been known since the late 1940s, neuronal dendrites have been considered as passive cables transferring incoming synaptic activity to the soma. The relatively recent discovery that neuronal dendrites contain active conductances has revolutionized our view of information processing in neurons. In many neuronal cell types, sodium action potentials initiated at the axon initial segment can back-propagate actively into the dendrite thereby serving, for the dendrite, as an indicator of the output activity of the neuron. In addition, the dendrites themselves can initiate action-potential-like regenerative responses, so-called dendritic spikes, that are mediated either by the activation of sodium, calcium, and/or N-methyl-D-aspartate receptor channels. Here, we review the recent experimental and theoretical evidence for a role of regenerative dendritic activity in information processing within neurons and, especially, in activity-dependent synaptic plasticity.     

Action potentials and chemosensitive conductances in the dendrites of olfactory neurons suggest new features for odor transduction

Odors affect the excitability of an olfactory neuron by altering membrane conductances at the ciliated end of a single, long dendrite. One mechanism to increase the sensitivity of olfactory neurons to odorants would be for their dendrites to support action potentials. We show for the first time that isolated olfactory dendrites from the mudpuppy Necturus maculosus contain a high density of voltage-activated Na+ channels and produce Na-dependent action potentials in response to depolarizing current pulses. Furthermore, all required steps in the transduction process beginning with odor detection and culminating with action potential initiation occur in the ciliated dendrite. We have previously shown that odors can modulate Cl- and K+ conductances in intact olfactory neurons, producing both excitation and inhibition. Here we show that both conductances are also present in the isolated, ciliated dendrite near the site of odor binding, that they are modulated by odors, and that they affect neuronal excitability. Voltage- activated Cl- currents blocked by 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid and niflumic acid were found at greater than five times higher average density in the ciliated dendrite than in the soma, whereas voltage-activated K+ currents inhibited by intracellular Cs+ were distributed on average more uniformly throughout the cell. When ciliated, chemosensitive dendrites were stimulated with the odorant taurine, the responses were similar to those seen in intact cells: Cl- currents were increased in some dendrites, whereas in others Cl- or K+ currents were decreased, and responses washed out during whole-cell recording. The Cl- equilibrium potential for intact neurons bathed in physiological saline was found to be -45 mV using an on-cell voltage- ramp protocol and delayed application of channel blockers. We postulate that transduction of some odors is caused by second messenger-mediated modulation of the resting membrane conductance (as opposed to a specialized generator conductance) in the cilia or apical region of the dendrite, and show how this could alter the firing frequency of olfactory neurons.     

Emerging methodologies for the study of hypothalamic gonadotropin-releasing-hormone (GnRH) neurons

Gonadotropin-releasing-hormone (GnRH) neurons form part of a central neural oscillator that controls sexual reproduction through intermittent release of the GnRH peptide. Activity of GnRH neurons, and by extension release of GnRH, has been proposed to reflect intrinsic properties and synaptic input of GnRH neurons. To study GnRH neurons, we used traditional electrophysiology and computational methods. These emerging methodologies enhance the elucidation of processing in GnRH neurons. We used dynamic current-clamping to understand how living GnRH somata process input from glutamate and GABA, two key neurotransmitters in the neuroendocrine hypothalamus. In order to study the impact of synaptic integration in dendrites and neuronal morphology, we have developed full-morphology models of GnRH neurons. Using dynamic clamping, we have demonstrated that small-amplitude glutamatergic currents can drive repetitive firing in GnRH neurons. Furthermore, application of simulated GABAergic synapses with a depolarized reversal potential have revealed two functional subpopulations of GnRH neurons: one population in which GABA chronically depolarizes membrane potential (without inducing action potentials) and a second population in which GABAergic excitation results in slow spiking. Finally, when AMPA-type and GABA-type simulated inputs are applied together, action potentials occur when the AMPA-type conductance occurs during the descending phase of GABAergic excitation and at the nadir of GABAergic inhibition. Compartmental computer models have shown that excitatory synapses at >300 microns from somtata are unable to drive spiking with purely passive dendrites. In models with active dendrites, distal synapses are more efficient at driving spiking than somatic inputs. We then used our models to extend the results from dynamic current clamping at GnRH somata to distribute synaptic inputs along the dendrite. We show that propagation delays for dendritic synapses alter synaptic integration in GnRH neurons by widening the temporal window of interaction for the generation of action potentials. Finally, we have shown that changes in dendrite morphology can modulate the output of GnRH neurons by altering the efficacy of action potential generation in response to after-depolarization potentials (ADPs). Taken together, the methodologies of dynamic current clamping and multi-compartmental modeling can make major contributions to the study of synaptic integration and structure-function relationships in hypothalamic GnRH neurons. Use of these methodological approaches will continue to provide keen insights leading to conceptual advances in our understanding of reproductive hormone secretion in normal and pathological physiology and open the door to understanding whether the mechanisms of pulsatile GnRH release are conserved across species.     

A unified model for two modes of bursting in GnRH neurons

Gonadotropin-releasing hormone (GnRH) neurons exhibit at least two intrinsic modes of action potential burst firing, referred to as parabolic and irregular bursting. Parabolic bursting is characterized by a slow wave in membrane potential that can underlie periodic clusters of action potentials with increased interspike interval at the beginning and at the end of each cluster. Irregular bursting is characterized by clusters of action potentials that are separated by varying durations of interburst intervals and a relatively stable baseline potential. Based on recent studies of isolated ionic currents, a stochastic Hodgkin-Huxley (HH)-like model for the GnRH neuron is developed to reproduce each mode of burst firing with an appropriate set of conductances. Model outcomes for bursting are in agreement with the experimental recordings in terms of interburst interval, interspike interval, active phase duration, and other quantitative properties specific to each mode of bursting. The model also shows similar outcomes in membrane potential to those seen experimentally when tetrodotoxin (TTX) is used to block action potentials during bursting, and when estradiol transitions cells exhibiting slow oscillations to irregular bursting mode in vitro. Based on the parameter values used to reproduce each mode of bursting, the model suggests that GnRH neurons can switch between the two through changes in the maximum conductance of certain ionic currents, notably the slow inward Ca²⁺ current I _s, and the Ca²⁺ -activated K⁺ current I _KCa. Bifurcation analysis of the model shows that both modes of bursting are similar from a dynamical systems perspective despite differences in burst characteristics.     

Effects of active conductance distribution over dendrites on the synaptic integration in an identified nonspiking interneuron

The synaptic integration in individual central neuron is critically affected by how active conductances are distributed over dendrites. It has been well known that the dendrites of central neurons are richly endowed with voltage- and ligand-regulated ion conductances. Nonspiking interneurons (NSIs), almost exclusively characteristic to arthropod central nervous systems, do not generate action potentials and hence lack voltage-regulated sodium channels, yet having a variety of voltage-regulated potassium conductances on their dendritic membrane including the one similar to the delayed-rectifier type potassium conductance. It remains unknown, however, how the active conductances are distributed over dendrites and how the synaptic integration is affected by those conductances in NSIs and other invertebrate neurons where the cell body is not included in the signal pathway from input synapses to output sites. In the present study, we quantitatively investigated the functional significance of active conductance distribution pattern in the spatio-temporal spread of synaptic potentials over dendrites of an identified NSI in the crayfish central nervous system by computer simulation. We systematically changed the distribution pattern of active conductances in the neuron's multicompartment model and examined how the synaptic potential waveform was affected by each distribution pattern. It was revealed that specific patterns of nonuniform distribution of potassium conductances were consistent, while other patterns were not, with the waveform of compound synaptic potentials recorded physiologically in the major input-output pathway of the cell, suggesting that the possibility of nonuniform distribution of potassium conductances over the dendrite cannot be excluded as well as the possibility of uniform distribution. Local synaptic circuits involving input and output synapses on the same branch or on the same side were found to be potentially affected under the condition of nonuniform distribution while operation of the major input-output pathway from the soma side to the one on the opposite side remained the same under both conditions of uniform and nonuniform distribution of potassium conductances over the NSI dendrite.     

Integration of subthreshold and suprathreshold excitatory barrages along the somatodendritic axis of pyramidal neurons

Neurons integrate inputs arriving in different cellular compartments to produce action potentials that are transmitted to other neurons. Because of the voltage- and time-dependent conductances in the dendrites and soma, summation of synaptic inputs is complex. To examine summation of membrane potentials and firing rates, we performed whole-cell recordings from layer 5 cortical pyramidal neurons in acute slices of the rat's somatosensory cortex. We delivered subthreshold and suprathreshold stimuli at the soma and several sites on the apical dendrite, and injected inputs that mimic synaptic barrages at individual or distributed sites. We found that summation of subthreshold potentials differed from that of firing rates. Subthreshold summation was linear when barrages were small but became supralinear as barrages increased. When neurons were discharging repetitively the rules were more diverse. At the soma and proximal apical dendrite summation of the evoked firing rates was predominantly sublinear whereas in the distal dendrite summation ranged from supralinear to sublinear. In addition, the integration of inputs delivered at a single location differed from that of distributed inputs only for suprathreshold responses. These results indicate that convergent inputs onto the apical dendrite and soma do not simply summate linearly, as suggested previously, and that distinct presynaptic afferents that target specific sites on the dendritic tree may perform unique sets of computations.     

Voltage-gated potassium currents are targets of diurnal changes in estradiol feedback regulation and kisspeptin action on gonadotropin-releasing hormone neurons in mice

Estradiol has both negative and positive feedback actions upon gonadotropin-releasing hormone (GnRH) release; the latter actions trigger the preovulatory GnRH surge. Although neurobiological mechanisms of the transitions between feedback modes are becoming better understood, the roles of voltage-gated potassium currents, major contributors to neuronal excitability, are unknown. Estradiol alters two components of potassium currents in these cells: a transient current, I(A), and a sustained current, I(K). Kisspeptin is a potential mediator between estradiol and GnRH neurons and can act directly on GnRH neurons. We examined how estradiol, time of day, and kisspeptin interact to regulate these conductances in a mouse model exhibiting daily switches between estradiol negative (morning) and positive feedback (evening). Whole-cell voltage clamp recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice and from OVX mice treated with estradiol (OVX+E). There were no diurnal changes in either I(A) or I(K) in GnRH neurons from OVX mice. In contrast, in GnRH neurons from OVX+E mice, I(A) and I(K) were greater during the morning when GnRH neuron activity is low and smaller in the evening when GnRH neuron activity is high. Estradiol increased I(A) in the morning and decreased it in the evening, relative to that in cells from OVX mice. Exogenously applied kisspeptin reduced I(A) regardless of time of day or estradiol status. Estradiol, interacting with time of day, and kisspeptin both depolarized I(A) activation. These findings extend our understanding of both the neurobiological mechanisms of estradiol negative vs. positive regulation of GnRH neurons and of kisspeptin action on these cells.     

Voltage-activated and odor-modulated conductances in olfactory neurons of Drosophila melanogaster

Voltage-activated currents and odor-modulated conductances were studied in cells in semi-intact Drosophila third antennal segments (the main olfactory organ) using patch-clamp techniques. All neurons expressed outward currents, and most expressed labile fast transient inward currents with kinetics similar to Na⁺ currents in other systems. Action potentials were detected as bipolar capacitative current transients in cell-attached or loose patches from the soma of both odor-sensitive (97%) and insensitive neurons. A mixture of odorants from five chemical classes caused an increase (∼70%), decrease (∼10%), or no effect on firing frequency in pharate adult neurons. The development of chemosensitivity was examined and odor-induced changes in action potential firing frequency were recorded in pupal antennal neurons as early as P8, a stage after completion of sensillar development. The character of odor-induced responses was more profound and complex later in development; small, tonic increases in firing frequency were observed at pupal stages P8 through P11(ii), while in older pupae and young adults ∼25% of the increased responses were phasic-tonic. The apical dendrite was the site of odor modulation in ∼90% and 100% of responsive adult and early pupal neurons, respectively. Whole-cell recordings revealed that apparent nonselective cation and chloride conductances were modulated by a mixture of odorants in separate antennal neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 123–137, 1997.     

Model of oscillatory activity in thalamic neurons: Role of voltage- and calcium-dependent ionic conductances

This paper describes a computer modeling study of the generation of 10 Hz oscillations in the electrical activity of guinea pig thalamic neurons in vitro. The computer model was based on experimental evidence suggesting that single thalamic neurons in guinea pig have a set of voltage- and calcium-dependent ionic conductances that is capable of generating self-sustained rhythmic oscillations. Simulation results are consistent with this hypothesis, and indicate that a model that contains dendritic calcium and calcium-dependent potassium conductances, as well as a voltage-dependent, slow sodium conductance, can indeed generate self-sustained oscillations like those seen in thalamic neurons. Moreover, simulations indicate that the occurrence of such oscillatory activity is strongly dependent on the location of the slow sodium conductance. Results predict that this slow sodium conductance is located in the dendrites.The authors express their appreciation to R. J. MacGregor for providing equations and computer programs for simulating a two-point neuronal model with active calcium-related conductances     

Diversity and modulation of ionic conductances in leech neurons

A complete understanding of animal behavior at the cellular level requires detailed information on the intrinsic biophysical properties of neurons, muscles, and the synaptic connections they make. In the past 10 to 15 years, electrophysiological studies of leech neurons have revealed a diverse array of voltage-gated ionic conductances distinguished by their pharmacological sensitivity to classic ion channel blockers. Voltage-clamp studies have provided new information about the kinetics and voltage-dependence of Na⁺ conductances, several K⁺ currents, including I_A, I_K and I_K(Ca.)' and high- and low-voltage-gated Ca²⁺ conductances. These studies showed that the action potentials of most leech neurons result from the usual sequence of permeability changes to Na⁺, K⁺, and Ca²⁺ ions. They also added insight as to the role played by particular combinations of conductances in providing individual neurons with electrical properties appropriate for the particular information they encode. Evidence is accumulating on the modulatory actions of endogenous neurotransmitters such as FMRFamide, serotonin, and octopamine on motor behaviors in the animal. Parallel studies suggest that changes in behavior can be explained, at least in part, by the alteration of firing patterns of selected neurons and muscles resulting form modulation of multiple ion conductances. This makes the leech exceptionally attractive for neuroethological studies because it is one of the simplest organisms in which the methods of psychology and neurobiology can be combined. Information gathered from this animal will therefore increase our understanding regarding general principles underlying the cellular basis of behavior. © 1995 John Wiley & Sons, Inc.     

Hyperforin attenuates various ionic conductance mechanisms in the isolated hippocampal neurons of rat

Effects of hyperforin, an acylphloroglucinol derivative isolated from antidepressive medicinal herb Hypericum perforatum (St. John's Wort), on voltage- and ligand-gated ionic conductances were investigated. Whole-cell patch clamp and concentration clamp techniques on acutely isolated hippocampal pyramidal neurons and on cerebellar Purkinje neurons of rat were used. At concentrations between 3 to 100 microM hyperforin induced a dose and time dependent inward current which completely stabilized within a few seconds. Although 1 microM hyperforin inhibited virtually all investigated conductances (GABA > or = I(Ca(N)) > I(Na) > I(Ca(P) > or = AMPA > or = I(K(A)) > NMDA > I(K(DR))), its effects on several of them could not be reversed by repeated washings. Dose response studies revealed that although AMPA induced current is inhibited by hyperforin in a competitive manner, these responses are not completely blocked by very high concentration of the agent. On the contrary, however, NMDA receptor-activated ionic conductance could be completely and uncompetitively inhibited by the agent. Taken together these observation not only reconfirm that hyperforin is a major neuroactive component of hypericum extracts but also demonstrate that this structurally unique and naturally abundant molecule is a potent modulation of mechanism involved in the control of neuronal ionic conductances. Various observed effects of hyperforin do not, however, seem to be mediated by one single molecular mechanism of action of the agent.     

Some aspects of the physiological role of ion channels in the nervous system

Recent analyses of the genomes of several animal species, including man, have revealed that a large number of ion channels are present in the nervous system. Our understanding of the physiological role of these channels in the nervous system has followed the evolution of biophysical techniques during the last century. The observation and the quantification of the electrical events associated with the operation of the ionic channels has been, and still is, one of the best tools to analyse the various aspects of their contribution to nerve function. For this reason, we have chosen to use electrophysiological recordings to illustrate some of the main functions of these channels. The properties and the roles of Na+ and K+ channels in neuronal resting and action potentials are illustrated in the case of the giant axons of the squid and the cockroach. The nature and role of the calcium currents in the bursting behaviour of the neurons are illustrated for Aplysia giant neurons. The relationship between presynaptic calcium currents and synaptic transmission is shown for the squid giant synapse. The involvement of calcium channels in survival and neurite outgrowth of cultured neurons is exemplified using embryonic cockroach brain neurons. This same neuronal preparation is used to illustrate ion channel noise and single-channel events associated with the binding of agonists to nicotinic receptors. Some features of the synaptic activity in the central nervous system are shown, with examples from the cercal nerve giant-axon preparation of the cockroach. The interplay of different ion conductances involved in the oscillatory behaviour of the Xenopus spinal motoneurons is illustrated and discussed. The last part of this review deals with ionic homeostasis in the brain and the function of glial cells, with examples from Necturus and squids.     

Whole-cell electrophysiology of gonadotropin-releasing hormone neurons that express green fluorescent protein in the terminal nerve of transgenic medaka (Oryzias latipes)

Gonadotropin-releasing hormone (GnRH) controls reproduction in vertebrates. Most studies have focused on the population of GnRH neurons in the hypothalamus that ultimately controls gonadal function. However, all vertebrates studied to date have two to three anatomically distinct populations of GnRH neurons that express different forms of this hormone. The purpose of the present study was to develop a new model for studying the population of GnRH neurons in the terminal nerve (TN) associated with the olfactory bulb and then to characterize their pattern of action potential firing to provide a foundation for understanding the role of these neurons in regulating reproduction. A stable line of transgenic medaka (Oryzias latipes) was generated in which a DNA construct containing the salmon GnRH (Gnrh3) promoter linked to green fluorescent protein (GFP) was expressed in TN-GnRH3 neurons. This population of GnRH neurons is located at or near the ventral surface of the brain, making them ideally situated for electrophysiological analysis. Whole-cell and loose-patch recordings in current-clamp mode were performed on these neurons from excised, intact brains of adult males in which afferent and efferent neural connections remained intact. All TN-GnRH3-GFP neurons that we recorded showed a beating pattern of spontaneous action potential firing. Action potentials were blocked by tetrodotoxin, indicating they are generated by a voltage-sensitive Na+ current; however, an oscillation in subthreshold membrane potential persisted. The present results indicate that this transgenic fish will provide an excellent model for studying the cell physiology of an extrahypothalamic population of GnRH neurons.     

A study on co-localization of FSH and its receptor in rat hippocampus

It has been known that GnRH, LH and their receptors exist in hippocampal neurons. However, whether FSH and its receptor also exist in hippocampal neurons remained unknown yet. In situ hybridization, double-labeled immunofluorescence stain and double-labeled immunohistochemistry stain in adjacent sections were used in our research to study the distribution, co-localization of FSH and its receptor and co-localization of FSH and GnRH receptor in rat hippocampus. The result found that pyramidal neurons from CA1 to CA4 region and granule neurons in dentate gyrus could express FSH and its receptor, majority of hippocampal neurons co-expressed FSH and its receptor, FSH and GnRH receptor. These suggested that hippocampal neurons not only express FSH but also act as FSH target cells. FSH may regulate the function of hippocampal neurons by ways of paracrine or autocrine. At the same time, GnRH may regulate the function of FSH neuron in hippocampus through GnRH receptor.     

Effects of exercise training on alpha-motoneurons.

Evidence is presented that one locus of adaptation in the "neural adaptations to training" is at the level of the alpha-motoneurons. With increased voluntary activity, these neurons show evidence of dendrite restructuring, increased protein synthesis, increased axon transport of proteins, enhanced neuromuscular transmission dynamics, and changes in electrophysiological properties. The latter include hyperpolarization of the resting membrane potential and voltage threshold, increased rate of action potential development, and increased amplitude of the afterhyperpolarization following the action potential. Many of these changes demonstrate intensity-related adaptations and are in the opposite direction under conditions in which chronic activity is reduced. A five-compartment model of rat motoneurons that innervate fast and slow muscle fibers (termed "fast" and "slow" motoneurons in this paper), including 10 active ion conductances, was used to attempt to reproduce exercise training-induced adaptations in electrophysiological properties. The results suggest that adaptations in alpha-motoneurons with exercise training may involve alterations in ion conductances, which may, in turn, include changes in the gene expression of the ion channel subunits, which underlie these conductances. Interestingly, the acute neuromodulatory effects of monoamines on motoneuron properties, which would be a factor during acute exercise as these monoaminergic systems are activated, appear to be in the opposite direction to changes measured in endurance-trained motoneurons that are at rest. It may be that regular increases in motoneuronal excitability during exercise via these monoaminergic systems in fact render the motoneurons less excitable when at rest. More research is required to establish the relationships between exercise training, resting and exercise motoneuron excitability, ion channel modulation, and the effects of neuromodulators.